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The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.
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Carcinoma de Pulmón de Células no Pequeñas , Daño del ADN , Histonas , Neoplasias Pulmonares , Organoides , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Organoides/metabolismo , Organoides/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Histonas/metabolismo , Línea Celular Tumoral , Imagen Óptica/métodos , Roturas del ADN de Doble CadenaRESUMEN
Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation, and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor-on-chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a HUVEC-derived vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells migrate; and a chemoattractant/tumor (HCC0827 or NCI-H520) compartment. Here, activated T-cells extravasate and migrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose translational strategies by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.
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Neoplasias Pulmonares , Sistemas Microfisiológicos , Animales , Humanos , Células Cultivadas , Endotelio Vascular , Neoplasias Pulmonares/tratamiento farmacológico , Movimiento CelularRESUMEN
We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
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Desarrollo de Medicamentos , Colorantes Fluorescentes/farmacología , Ensayos Analíticos de Alto Rendimiento , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Estructura Molecular , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-ActividadRESUMEN
SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.
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Dendritas/fisiología , Red Nerviosa/fisiología , Sistema Nervioso/crecimiento & desarrollo , Sinapsis/fisiología , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/fisiología , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Tamaño de la Célula , Células Cultivadas , Potenciales Postsinápticos Excitadores/genética , Femenino , Eliminación de Gen , Humanos , Trastornos del Neurodesarrollo/genética , Células Madre PluripotentesRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related deaths in the United States by 2020, due in part to innate resistance to widely used chemotherapeutic agents and limited knowledge about key molecular factors that drive tumor aggression. We previously reported a novel negative prognostic biomarker, keratin 17 (K17), whose overexpression in cancer results in shortened patient survival. In this study, we aimed to determine the predictive value of K17 and explore the therapeutic vulnerability in K17-expressing PDAC, using an unbiased high-throughput drug screen. Patient-derived data analysis showed that K17 expression correlates with resistance to gemcitabine (Gem). In multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells, and orthotopic xenografts, we determined that the expression of K17 results in a more than twofold increase in resistance to Gem and 5-fluorouracil, key components of current standard-of-care chemotherapeutic regimens. Furthermore, through an unbiased drug screen, we discovered that podophyllotoxin (PPT), a microtubule inhibitor, showed significantly higher sensitivity in K17-positive compared to K17-negative PDAC cell lines and animal models. In the clinic, another microtubule inhibitor, paclitaxel (PTX), is used in combination with Gem as a first-line chemotherapeutic regimen for PDAC. Surprisingly, we found that when combined with Gem, PPT, but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells. Importantly, in preclinical models, PPT in combination with Gem effectively decreased tumor growth and enhanced the survival of mice bearing K17-expressing tumors. This provides evidence that PPT and its derivatives could potentially be combined with Gem to enhance treatment efficacy for the ~ 50% of PDACs that express high levels of K17. In summary, we reported that K17 is a novel target for developing a biomarker-based personalized treatment for PDAC.
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Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Humanos , Queratina-17/metabolismo , Ratones Endogámicos C57BL , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/patología , Podofilotoxina/farmacología , Podofilotoxina/uso terapéutico , Carga Tumoral/efectos de los fármacos , GemcitabinaRESUMEN
Neurons created from human induced pluripotent stem cells (hiPSCs) provide the capability of identifying biological mechanisms that underlie brain disorders. IPSC-derived human neurons, or iNs, hold promise for advancing precision medicine through drug screening, though it remains unclear to what extent iNs can support early-stage drug discovery efforts in industrial-scale screening centers. Despite several reported approaches to generate iNs from iPSCs, each suffer from technological limitations that challenge their scalability and reproducibility, both requirements for successful screening assays. We addressed these challenges by initially removing the roadblocks related to scaling of iNs for high throughput screening (HTS)-ready assays. We accomplished this by simplifying the production and plating of iNs and adapting them to a freezer-ready format. We then tested the performance of freezer-ready iNs in an HTS-amenable phenotypic assay that measured neurite outgrowth. This assay successfully identified small molecule inhibitors of neurite outgrowth. Importantly, we provide evidence that this scalable iN-based assay was both robust and highly reproducible across different laboratories. These streamlined approaches are compatible with any iPSC line that can produce iNs. Thus, our findings indicate that current methods for producing iPSCs are appropriate for large-scale drug-discovery campaigns (i.e. >10e5 compounds) that read out simple neuronal phenotypes. However, due to the inherent limitations of currently available iN differentiation protocols, technological advances are required to achieve similar scalability for screens that require more complex phenotypes related to neuronal function.
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Diferenciación Celular/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Células Madre Pluripotentes Inducidas/fisiología , Neuronas/fisiología , Bioensayo/métodos , Células Cultivadas , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Proyección Neuronal/efectos de los fármacos , Proyección Neuronal/fisiología , Neuronas/citología , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
INTRODUCTION: Spheroids of mesenchymal stem cells (MSCs) in cartilage tissue engineering have been shown to enhance regenerative potential owing to their 3D structure. In this study, we explored the possibility of priming spheroids under different media to replace the use of inductive surface coatings for chondrogenic differentiation. METHODS: Rat bone marrow-derived MSCs were organized into cell spheroids by the hanging drop technique and subsequently cultured on hyaluronic acid (HA) coated or non-coated well plates under different cell media conditions. Endpoint analysis included cell viability, DNA and Glycosaminoglycan (GAG) and collagen content, gene expression and immunohistochemistry. RESULTS: For chondrogenic applications, MSC spheroids derived on non-coated surfaces outperformed the spheroids derived from HA-coated surfaces in matrix synthesis and collagen II gene expression. Spheroids on non-coated surfaces gave rise to the highest collagen and GAG when primed with medium containing insulin-like growth factor (IGF) for 1 week during spheroid formation. Spheroids that were grown in chondroinductive raw material-inclusive media such as aggrecan or chondroitin sulfate exhibited the highest Collagen II gene expression in the non-coated surface at 1 week. CONCLUSION: Media priming by growth factors and raw materials might be a more predictive influencer of chondrogenesis compared to inductive-surfaces. Such tailored bioactivity of the stem cell spheroids in the stage of the spheroid formation may give rise to a platform technology that may eventually produce spheroids capable of chondrogenesis achieved by mere media manipulation, skipping the need for additional culture on a modified surface, that paves the way for cost-effective technologies.
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To date, many osteochondral regenerative approaches have utilized varied combinations of biocompatible materials and cells to engineer cartilage. Even in cell-based approaches, to date, no study has utilized stem cell aggregates alone for regenerating articular cartilage. Thus, the purpose of this study was to evaluate the performance of a novel stem cell-based aggregate approach in a fibrin carrier to regenerate osteochondral defects in the Sprague-Dawley rat trochlear groove model. Two different densities of rat bone marrow mesenchymal stem cell (rBMSC) aggregates were fabricated by the hanging drop technique. At 8 weeks, the cell aggregates supported the defects and served as a catalyst for neo-cartilage synthesis, and the experimental groups may have been beneficial for bone and cartilage regeneration compared to the fibrin-only control and sham groups, as evidenced by histological assessment. The cell density of rBMSC aggregates may thus directly impact chondrogenesis. The usage of cell aggregates with fibrin as a cell-based technology is a promising and translational new treatment strategy for repair of cartilage defects. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1606-1616, 2017.
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Cartílago Articular/fisiología , Trasplante de Células Madre Mesenquimatosas/métodos , Regeneración , Animales , Agregación Celular , Fibrina , Masculino , Proyectos Piloto , Cultivo Primario de Células , Ratas Sprague-DawleyRESUMEN
"Raw materials," or materials capable of serving both as building blocks and as signals, which are often but not always natural materials, are taking center stage in biomaterials for contemporary regenerative medicine. In osteochondral tissue engineering, a field leveraging the underlying bone to facilitate cartilage regeneration, common raw materials include chondroitin sulfate (CS) for cartilage and ß-tricalcium phosphate (TCP) for bone. Building on our previous work with gradient scaffolds based on microspheres, here we delved deeper into the characterization of individual components. In the current study, the release of CS and TCP from poly(D, L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds was evaluated over a time period of 4 weeks. Raw material encapsulated groups were compared to 'blank' groups and evaluated for surface topology, molecular weight, and mechanical performance as a function of time. The CS group may have led to increased surface porosity, and the addition of CS improved the mechanical performance of the scaffold. The finding that CS was completely released into the surrounding media by 4 weeks has a significant impact on future in vivo studies, given rapid bioavailability. The addition of TCP seemed to contribute to the rough external appearance of the scaffold. The current study provides an introduction to degradation patterns of homogenous raw material encapsulated scaffolds, providing characterization data to advance the field of microsphere-based scaffolds in tissue engineering.
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Fosfatos de Calcio/química , Sulfatos de Condroitina/química , Microesferas , Fosfatos de Calcio/metabolismo , Sulfatos de Condroitina/metabolismo , Módulo de Elasticidad , Ácido Láctico/química , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Peso Molecular , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porosidad , Propiedades de Superficie , Resistencia a la Tracción , Factores de TiempoRESUMEN
Despite numerous efforts in cartilage regeneration, few products see the light of clinical translation as the commercialization process is opaque, financially demanding, and requires collaboration with people of varied skill sets. The aim of this review is to introduce, to an academic audience, the different paradigms involved in the commercialization of cartilage regeneration technology, elucidate the different hurdles associated with the use of cells and materials in developing new technologies, discuss potential commercialization strategies, and inform the reader about the current trends observed in both the clinical and laboratory setting for establishing clinical trials. Although there are review articles on articular cartilage tissue engineering, independent reports provided by the Food and Drug Administration, and separate review articles on animal models, this is the first review that encompasses all of these facets and is presented in a format favorable to the academic investigator interested in clinical translation from bench to bedside.
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There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton's jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance.
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Condrogénesis , Hidrogeles , Células Madre/citología , Células Madre/fisiología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Colágeno/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratas , Cordón Umbilical/citologíaRESUMEN
Scaffolds with continuous gradients in material composition and bioactive signals enable a smooth transition of properties at the interface. Components like chondroitin sulfate (CS) and bioactive glass (BG) in 3D scaffolds may serve as "raw materials" for synthesis of new extracellular matrix (ECM), and may have the potential to completely or partially replace expensive growth factors. We hypothesized that scaffolds with gradients of ECM components would enable superior performance of engineered constructs. Raw material encapsulation altered the appearance, structure, porosity, and degradation of the scaffolds. They allowed the scaffolds to better retain their 3D structure during culture and provided a buffering effect to the cells in culture. Following seeding of rat mesenchymal stem cells, there were several instances where glycosaminoglycan (GAG), collagen, or calcium contents were higher with the scaffolds containing raw materials (CS or BG) than with those containing transforming growth factor (TGF)-ß3 or bone morphogenetic protein (BMP)-2. It was also noteworthy that a combination of both CS and TGF-ß3 increased the secretion of collagen type II. Moreover, cells seeded in scaffolds containing opposing gradients of CS/TGF-ß3 and BG/BMP-2 produced clear regional variations in the secretion of tissue-specific ECM. The study demonstrated raw materials have the potential to create a favorable microenvironment for cells; they can significantly enhance the synthesis of certain extracellular matrix (ECM) components when compared to expensive growth factors; either alone or in combination with growth factors they can enhance the secretion of tissue specific matrix proteins. Raw materials are promising candidates that can be used to either replace or be used in combination with growth factors. Success with raw materials in lieu of growth factors could have profound implications in terms of lower cost and faster regulatory approval for more rapid translation of regenerative medicine products to the clinic.
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Sulfatos de Condroitina/farmacocinética , Microesferas , Ingeniería de Tejidos/instrumentación , Andamios del Tejido , Animales , Células de la Médula Ósea , Regeneración Ósea , Condrocitos/citología , Condrocitos/fisiología , Portadores de Fármacos , Ensayo de Materiales , Ratas , Células MadreRESUMEN
The aim of this study was to use CO2 at sub-critical pressures as a tool to sinter 3D, macroporous, microsphere-based scaffolds for bone and cartilage tissue engineering. Porous scaffolds composed of ~200 µm microspheres of either poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) were prepared using dense phase CO2 sintering, which were seeded with rat bone marrow mesenchymal stromal cells (rBMSCs), and exposed to either osteogenic (PLGA, PCL) or chondrogenic (PLGA) conditions for 6 weeks. Under osteogenic conditions, the PLGA constructs produced over an order of magnitude more calcium than the PCL constructs, whereas the PCL constructs had far superior mechanical and structural integrity (125 times stiffer than PLGA constructs) at week 6, along with twice the cell content of the PLGA constructs. Chondrogenic cell performance was limited in PLGA constructs, perhaps as a result of the polymer degradation rate being too high. The current study represents the first long-term culture of CO2-sintered microsphere-based scaffolds, and has established important thermodynamic differences in sintering between the selected formulations of PLGA and PCL, with the former requiring adjustment of pressure only, and the latter requiring the adjustment of both pressure and temperature. Based on more straightforward sintering conditions and more favorable cell performance, PLGA may be the material of choice for microspheres in a CO2 sintering application, although a different PLGA formulation with the encapsulation of growth factors, extracellular matrix-derived nanoparticles, and/or buffers in the microspheres may be advantageous for achieving a more superior cell performance than observed here.
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Materiales Biocompatibles/química , Dióxido de Carbono/química , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Ingeniería de Tejidos , Animales , Materiales Biocompatibles/farmacología , Células de la Médula Ósea/citología , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Condrogénesis/efectos de los fármacos , Módulo de Elasticidad , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glicosaminoglicanos/análisis , Hidroxiprolina/análisis , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Nanopartículas/química , Osteogénesis/efectos de los fármacos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porosidad , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Andamios del TejidoRESUMEN
Microsphere-based polymeric tissue-engineered scaffolds offer the advantage of shape-specific constructs with excellent spatiotemporal control and interconnected porous structures. The use of these highly versatile scaffolds requires a method to sinter the discrete microspheres together into a cohesive network, typically with the use of heat or organic solvents. We previously introduced subcritical CO(2) as a sintering method for microsphere-based scaffolds; here we further explored the effect of processing parameters. Gaseous or subcritical CO(2) was used for making the scaffolds, and various pressures, ratios of lactic acid to glycolic acid in poly(lactic acid-co-glycolic acid), and amounts of NaCl particles were explored. By changing these parameters, scaffolds with different mechanical properties and morphologies were prepared. The preferred range of applied subcritical CO(2) was 15-25 bar. Scaffolds prepared at 25 bar with lower lactic acid ratios and without NaCl particles had a higher stiffness, while the constructs made at 15 bar, lower glycolic acid content, and with salt granules had lower elastic moduli. Human umbilical cord mesenchymal stromal cells (hUCMSCs) seeded on the scaffolds demonstrated that cells penetrate the scaffolds and remain viable. Overall, the study demonstrated the dependence of the optimal CO(2) sintering parameters on the polymer and conditions, and identified desirable CO(2) processing parameters to employ in the sintering of microsphere-based scaffolds as a more benign alternative to heat-sintering or solvent-based sintering methods.
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Andamios del Tejido , Fenómenos Biomecánicos , Dióxido de Carbono , Supervivencia Celular , Células Cultivadas , Humanos , Ácido Láctico , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Microscopía Electrónica de Rastreo , Microesferas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porosidad , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Cordón Umbilical/citologíaRESUMEN
Pathogenic agents can lead to severe clinical outcomes such as food poisoning, infection of open wounds, particularly in burn injuries and sepsis. Rapid detection of these pathogens can monitor these infections in a timely manner improving clinical outcomes. Conventional bacterial detection methods, such as agar plate culture or polymerase chain reaction, are time-consuming and dependent on complex and expensive instruments, which are not suitable for point-of-care (POC) settings. Therefore, there is an unmet need to develop a simple, rapid method for detection of pathogens such as Escherichia coli. Here, we present an immunobased microchip technology that can rapidly detect and quantify bacterial presence in various sources including physiologically relevant buffer solution (phosphate buffered saline [PBS]), blood, milk, and spinach. The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein. The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively. The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds.
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Escherichia coli/aislamiento & purificación , Microbiología de Alimentos/instrumentación , Microbiología de Alimentos/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Animales , Anticuerpos Inmovilizados/metabolismo , Sangre/microbiología , Recuento de Colonia Microbiana , Escherichia coli/metabolismo , Humanos , Leche/microbiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Spinacia oleracea/microbiología , Estadísticas no Paramétricas , Propiedades de SuperficieRESUMEN
Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method.
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Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types.